Viroides, pequenos RNAs patogênicos capazes de replicação autônoma: modelos moleculares para o estudo de interações patógeno-hospedeiro e evolução / Viroids, small pathogenic RNAs capable of autonomous replication: molecular models for the study on host-pathogen interactions and evolution

Os viroides, apesar de serem constituídos por um pequeno RNA de fita simples, fortemente estruturado, circular, que não codifica proteínas, são capazes de se replicar de maneira autônoma em plantas superiores e causar doença interagindo diretamente com fatores do hospedeiro. Nesta revisão, serão apresentados e discutidos alguns dos mais recentes trabalhos envolvendo a interação de viroides com fatores do hospedeiro, incluindo aspectos relacionados à replicação, movimento e patogênese, além de suas características evolutivas. Nos últimos anos, alguns grupos de pesquisa têm se aventurado na busca por fatores do hospedeiro e mecanismos moleculares relacionados ao ciclo infeccioso dos viroides, tentando desvendar como esses pequenos RNAs interagem com o hospedeiro induzindo sintomas. Os viroides não codificam proteínas supressoras de silenciamento e, portanto, devem garantir sua existência utilizando estratégias baseadas em sua estrutura secundária, na compartimentalização em organelas, associação com fatores do hospedeiro e eficiência na replicação. A complexidade do ciclo infeccioso desses minúsculos RNAs indica que muitas interações desses patógenos com fatores do hospedeiro ainda devem ser identificadas.

Viroids are small, single-stranded, highly structured, circular RNAs that replicate autonomously in their hosts, without messenger RNA activity. Because they do not encode for proteins, viroids have to interact directly with host factors. This review presents recent progress in understanding the possible role of recently identified viroid-binding host proteins related to replication, trafficking and pathogenesis. It also discusses some aspects on viroid evolution. In recent years, efforts to understand how viroids replicate, cause disease and induce symptoms have prompted details on molecular mechanisms related to the viroid infectious cycle. Inasmuch as viroids lack protein-encoding capacity, including suppressors of gene silencing, their existence could be ensured by their compact conformation, compartimentalization in organelles, association with host factors or by their highly efficient replication. The complexity of the infectious cycle of these tiny pathogenic RNAs indicates that several interactions with host factors remain to be identified.

Analysis of viroid replication.

Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246-401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infected plants, transcription assays with nuclear and chloroplastic fractions, characterization of enzymes and ribozymes mediating cleavage and ligation of viroid strands, dissection of 5' terminal groups of viroid strands, and in situ hybridization and microscopy of subcellular fractions and tissues), and in vivo systems (tissue infiltration studies, protoplasts, studies in planta and use of transgenic plants expressing viroid RNAs).

Deletion analysis of the sapovirus VP1 gene for the assembly of virus-like particles.

Human sapovirus (SaV) strains are agents of gastroenteritis. They cannot be grown in cell culture. In this study, constructs containing SaV N- and C-terminal-deleted recombinant capsid proteins (rVP1) were expressed in a baculovirus expression system to allow us to better understand the sequence requirements for the formation of virus-like particles (VLPs). Only proteins derived from N-terminal-deleted rVP1 constructs that began 49 nucleotides downstream assembled into VLPs, which included both small and native-size VLPs. Our results were similar to those reported in a rabbit hemorrhagic disease virus (RHDV) N- and C-terminal-deleted rVP1 expression study but were distinct from those reported in a norovirus N- and C-terminal-deleted rVP1 expression study, suggesting that SaV and RHDV may have similar expression requirements.

In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket.

Peach latent mosaic viroid (PLMVd) can adopt hammerhead structures in both polarity strands. In the course of a study on the variability of this viroid a natural sequence variant has been characterized in which the hammerhead structure of the plus polarity strand has the sequence CCGA instead of the conserved uridine turn motif CUGA present in the catalytic pocket of all natural hammerhead structures. The viroid RNA containing this mutant hammerhead structure, but not those with the two other possible substitutions, U-->A and U-->G, in the same position of the catalytic pocket, showed significant self-cleavage activity during in vitro transcription. Moreover, the corresponding full-length PLMVd cDNA was infectious and the mutation was retained in a fraction of the viroid progeny. These results indicate that the sequence flexibility of the hammerhead structure, acting in vitro and in vivo , is higher than anticipated and provide relevant data for a deeper insight into the catalytic mechanism of this class of ribozymes and into the structure of the uridine turn motif.

Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch.

Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-linkable loop is closed and thus contains non-canonical base-pairs ("loop E structure"). (2) Three large "pre-melting loops" are present at 25 degrees to 37 degrees C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all "large" viroids (approximately 300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine lacks one of these hairpins, whereas all "small" viroids (approximately 300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other.

Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid.

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV-crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV-crosslinking between the same two bases as in full-length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.

Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy.

The intracellular localization of viroids has been investigated by viroid-specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine--rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC-conjugated antibody. In plants infected with either a lethal or an intermediate PSTVd strain, the highest intensity of fluorescence that arose from hybridization with the probe specific for the viroid (+)strand was found in the nucleoli, confirming results of previous fractionation studies. A similar distribution was found for (-)strand replication intermediates of PSTVd using specific (+)strand transcripts as hybridization probes. In order to determine if viroids are located at the surface or in the interior of the nucleoli, the distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM). It was shown by three-dimensional reconstruction that viroids are neither restricted to the surface of the nucleoli nor to a peripheral zone, but are instead homogeneously distributed throughout the nucleolus. The functional implications of the intranucleolar location of viroids and their replication intermediates are discussed with respect to proposed mechanisms of viroid replication and pathogenesis.

Two related viroids cause grapevine yellow speckle disease independently.

We have confirmed that two closely related circular RNA molecules previously named grapevine yellow speckel viroid (GYSV) and grapevine viroid 1B (GV1B) are indeed viroids. Electron microscopy after spreading under non-denaturing conditions revealed that GYSV has a rod-like structure typical of viroids. Purified GYSV and GV1B replicated independently in inoculated grapevine seedlings and some of the infected plants developed yellow speckle symptoms indicating that both viroids can cause grapevine yellow speckle disease. Plus-sense RNA transcripts derived from a dimeric GYSV cDNA clone induced yellow speckle symptoms in a grapevine seedling confirming the role of GYSV in the yellow speckle disease. Two oligonucleotide probes were synthesized for the detection of the two related viroids. The probes which could detect each viroid individually were used to assess correlations between the occurrence of these viroids and the incidence of the disease.

Grapevine yellow speckle viroid: structural features of a new viroid group.

A single stranded circular RNA was isolated from grapevines infected with yellow speckle disease. The RNA which we have called grapevine yellow speckle viroid (GYSV), contains 367 nucleotide residues and has the potential to form the rod-like secondary structure characteristic of viroids. GYSV has 37% sequence homology with the recently described apple scar skin viroid (ASSV; 330 residues) and has some sequence homology with the viroids in the potato spindle tuber viroid (PSTV) group. The sequence of GYSV has characteristics which fit the structural domains described for the PSTV group. However, GYSV lacks the PSTV central conserved sequence. Instead, there is a conserved sequence in the central region of GYSV and ASSV which has the potential to form a stem loop configuration and a stable palindromic structure as does the central conserved region of the PSTV group. These structural features suggest there is a different central conserved region for GYSV and ASSV. The results support the viroid nature of GYSV and its inclusion into a separate viroid group which we suggest should be represented by ASSV.

Complexes of viroids with histones and other proteins.

Complexes of potato spindle tuber viroid (PSTV) with nuclear proteins have been studied by in vitro reconstitution of the complexes and by isolation and characterization of in vivo complexes under non-dissociating conditions. For in vitro reconstitution, nuclear proteins were separated by SDS-gel-electrophoresis, renatured and blotted onto nitrocellulose filters, and incubated with viroid. The viroid-protein complexes were crosslinked covalently, and the viroid containing protein bands were detected by northern hybridization with a radioactive cDNA probe. The histones, a 41,000 dalton protein and to a small extent a 31,000 dalton protein were found in complexes with viroids. Raising the strength to 0.4 M NaCl destroys the complexes with the 41,000 dalton proteins but not those with the histones. From nucleoli, which are known to obtain the majority of viroids under non-dissociating conditions (Schumacher et al., (1983) EMBO J. 2, 1549-1555), a nucleosomal fraction was prepared. Viroids were found predominantly in this nucleosomal fraction. They are bound in a complex of 12-15 svedberg units.

Conformational transitions in viroids and virusoids: comparison of results from energy minimization algorithm and from experimental data.

Viroids are single-stranded circular RNA molecules of 240 to 400 nucleotides which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but replicate only as genomic part of a plant virus. Their structure and structural transitions have been investigated by thermo-dynamic, kinetic and hydrodynamic methods. The special features of the sequences of these RNAs, which are the basis for their secondary structures and structural flexibility, are investigated with theoretical methods. A set of thermodynamic parameters for helix growth and loop formation is selected from the literature to calculate secondary structures and structural transitions of single-stranded RNAs. Appropriate modifications of the chosen parameter set are discussed. For calculations we used either Tinoco-plots and the model of "cooperative helices" or the Zuker-program based on the exact algorithm of Nussinov et al, or both. Calculations were done for viroids and virusoids. As both are single-stranded, circular RNAs we had to modify the Zuker-program as described in the appendix. Calculations are done for different viroids, i.e. potato spindle tuber, citrus exocortis, chrysanthemum stunt, coconut cadang-cadang, and avocado sunblotch, and for two virusoids, i.e. the circular RNAs of Solanum nodiflorum mottle virus, and velvet tobacco mottle virus. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but statistical sequences. The comparison of viroids, virusoids, and circular RNA or random sequences confirms the uniqueness of viroid structure.

Dynamics and interactions of viroids.

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.

Demonstration of two different types of non-A, non-B hepatitis by reinjection and cross-challenge studies in chimpanzees.

Utilizing immune electron microscopy, viruslike particles were identified in the serums of three apparently healthy, HBsAg-negative blood donors. The serum from one of the donors, when injected into two susceptible chimpanzees, induced non-A, non-B hepatitis with an increase in SGPT level and pathological changes in the liver compatible with acute hepatitis but none of the cytoplasmic ultrastructures previously noted by electron microscopy in non-A, non-B hepatitis. These two chimps did not contract hepatitis when the same inoculum was given again 17 wk after the first injection. When they were subsequently challenged, however, by a chimp inoculum containing viruslike particles known to induce non-A, non-B hepatitis with cytoplasmic tubular ultrastructures, they developed high SGPT levels and these characteristic ultrastructures in their hepatocytes. A third chimp was initially injected with the chimp inoculum containing viruslike particles known to induce hepatitis with tubular ultrastructures, reinjected with the same agent, and then challenged by the human serum containing viruslike particles capable of inducing non-A, non-B hepatitis without tubular ultrastructures. He developed biochemical and pathological evidence of acute hepatitis after the first and the third, but not after the second inoculations. There are at least two kinds of viruslike particles which are associated with infectivity for two different types of non-A, non-B hepatitis; these have been tentatively designated NANB-1 and NANB-2.